Human Hookworm Vaccine

ABSTRACT

A vaccine for human hookworm is provided. The vaccine comprises at least one L3 larval stage antigen (e.g. Na-ASP-2 or Na-SAA-2) and at least one adult stage human hookworm antigen (e.g. Na-APR-1, Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or Na-GST-1) and adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. patent application Ser. No.10/825,692, filed Apr. 16, 2004, and to U.S. provisional patentapplication 60/862,916, filed Oct. 25, 2006, the complete contents ofboth of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to a vaccine for human hookworm. Inparticular, the invention provides a human hookworm vaccine comprisingan L3 larval stage antigen (e.g. Na-ASP-2 or Na-SAA-2) and at least oneadult stage human hookworm antigen (e.g. Na-APR-1, Na-CP-2, Na-CP-3,Na-CP-4, Na-CP-5, or Na-GST-1) and two or more adjuvants, one of whichis an aluminum-based adjuvant such as Alhydrogel®.

2. Background of the Invention

Hookworms are gastrointestinal nematodes that infect approximately 600million people in developing countries (Hotez et al, 2006a). Adulthookworms bury their heads beneath the mucosa of the human intestine andfeed on blood. Moderate to heavy infections result in iron deficiencyanaemia, the major pathologic sequella of hookworm disease, as well asprotein malnutrition. The resulting hookworm disease and anemia has aserious deleterious impact on many aspects of the health of infectedindividuals, including childhood growth retardation and cognitivedevelopment, and impaired fetal development during pregnancy (Hotez etal, 2004). The global disease burden resulting from chronic hookworminfection in childhood and pregnancy is enormous, possibly as high as 22million disability-adjusted life years annually (Chan, 1997), makinghookworm the second most important parasitic infection of humans aftermalaria (Hotez et al, 2005). In addition, the chronic immune suppressioninduced by hookworms and other helminths also has enormous impact on theability of people to respond in a competent fashion to other infections(including malaria and HIV/AIDS and vaccines (Elliott et al, 2005; Su etal. 2005; Cooper et al., 2001; Cooper et al., 1999; Hotez et al, 2006b).

Unlike many other human helminthiases, clear-cut immunity againsthookworms does not develop in the majority of infected individuals(Loukas et al., 2005). Indeed, the oldest people living in an endemiccommunity sometimes have the heaviest worm burdens (Bethony et al.,2002). While anthelminthic drugs of the benzimidazole class are highlyeffective at eliminating existing hookworm infections, they do notprotect against rapid re-infection (Hotez et al, 2006a). In areas ofhigh transmission, hookworm re-infection will occur within 4-12 months(Albonico et al, 1995), leading to concerns about the long-termsustainability of such practices (Kremer 2004). In addition, newer dataindicates that the efficacy of benzimidazole drugs decreases withfrequent use (Albonico et al, 2003), leading to concerns about thepossibility that anthelminthic drug resistance has developed (Albonicoet al, 2004; Bethony et al, 2006). These observations have led to callsby the World Health Organization and other international agencies todevelop new tools for the control of hookworm, including a hookwormvaccine (WHO, 2005). Therefore, an anthelminthic vaccine that inducesimmunological protection to minimize pathology and interrupt hookwormtransmission is a highly desirable goal.

While regional economic growth (and with it, improvements in sanitationand clean water) in some parts of North America, Japan, South Korea, andChina have translated into substantial reductions in endemic hookworm(Hotez et al, 2006a), estimated prevalence rates for the world's poorestand least developed regions remain high. For example, infection rates insub-Saharan Africa (SSA) are equivalent to those first estimated morethan 60 years ago (DeSilver et al., 2003), where an estimated 198million cases occur (DeSilva et al, 2003). High hookworm infestationrates are principally in poverty-stricken rural areas where access tomedical care is severely limited. Widespread use of a hookworm vaccinewould lead to significant improvement in global health and in economicdevelopment (Hotez et al, 2006a; Hotez and Ferris, 2006). Therefore, anideal vaccine hookworm vaccine would also be relatively easy andinexpensive to produce, and would be effective without the need forconstant boosting.

The prior art has thus far failed to provide such a vaccine againsthuman hookworm.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a bivalent human hookwormvaccine. The vaccine is effective at inducing an immune response inindividuals to whom it is administered, and administration results in areduction in symptoms of hookworm disease.

The vaccine comprises: one or more L3 larval stage antigen (e.g.Na-ASP-2 and/or Na-SAA-2) and at least one adult stage human hookwormantigen (e.g. Na-APR-1, Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or Na-GST-1)and one or more adjuvants. In some embodiments, the vaccine compositionincludes two or more adjuvants, one of which is an aluminum-basedadjuvant such as Alhydrogel®.

The present invention provides a hookworm vaccine comprising a hookwormlarval stage antigen, a hookworm adult stage antigen, and one or moreadjuvants. In one embodiment of the invention, the vaccine includes atleast one larval-stage hookworm antigen, at least one adult-stagehookworm antigen, an aluminum-based adjuvant, and a second adjuvant. Inone embodiment of the invention, the larval-stage hookworm antigen isNa-ASP-2 or Na-SAA-2, or both. Further, the larval-stage hookwormantigen may be antigenic fragments of Na-ASP-2 or Na-SAA-2, or both. Inone embodiment of the invention, the adult-stage hookworm antigen isNa-APR-1, Na-GST, Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or antigenicfragments thereof, or a combination of several of these antigens. In oneembodiment of the invention, the Na-APR-1 that is utilized is Pichiaoptimized Na-APR-1, or an antigenic fragment thereof. In someembodiments, the aluminum-based adjuvant is Alhydrogel® and the secondadjuvant is CpG or Synthetic lipid A. In some embodiments of theinvention, the aluminum-based adjuvant and the second adjuvant arecombined together.

The invention also includes a method for vaccinating a patient in needthereof against hookworm infections. The method comprises the step ofadministering to the patient a hookworm vaccine comprising a hookwormlarval stage antigen, a hookworm adult stage antigen, and one or moreadjuvants. In one embodiment of the invention, the vaccine includes atleast one larval-stage hookworm antigen, at least one adult-stagehookworm antigen, an aluminum-based adjuvant, and a second adjuvant. Inone embodiment of the invention, the larval-stage hookworm antigen isNa-ASP-2 or Na-SAA-2, or both. Further, the larval-stage hookwormantigen may be antigenic fragments of Na-ASP-2 or Na-SAA-2, or both. Inone embodiment of the invention, the adult-stage hookworm antigen isNa-APR-1, Na-GST, Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or antigenicfragments thereof, or a combination of several of these antigens. In oneembodiment of the invention, the Na-APR-1 that is utilized is Pichiaoptimized Na-APR-1, or an antigenic fragment thereof. In someembodiments, the aluminum-based adjuvant is Alhydrogel® and the secondadjuvant is CpG or Synthetic lipid A. In some embodiments of theinvention, the aluminum-based adjuvant and the second adjuvant arecombined together. In one embodiment, the method further comprises thestep of administering a deworming agent to said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-ASP-2.

FIGS. 2A and B. A, cDNA nucleotide sequence (partial sequence 62-1351bp); and B, amino acid sequence of Na-APR-1, Shanghai strain.

FIGS. 3A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceof Na-APR-1, Australian strain.

FIG. 4. Amino acid sequence (without signal sequence) alignment betweenShanghai and Australian strains of Na-APR-1.

FIGS. 5A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceof Pichia optimized Na-APR-1 (Na-APR-1-0), based on Australian strain;the sequence is identical to residues 17-446 of Na-APR-1 Australianstrain.

FIGS. 6A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Pichia optimized Na-APR-1 (Australianstrain) with Asp97 mutated to Ala97 (shown in bold and underlined).

FIGS. 7A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Pichia optimized Na-APR-1 (Australianstrain) with Asp284 mutated to Ala284.

FIGS. 8A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Pichia optimized Na-APR-1 (Australianstrain) with both Asp97 mutated to Ala97 and Asp284 mutated to Ala284.

FIGS. 9A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-GST-1.

FIGS. 10A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-GST-2.

FIGS. 11A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-GST-3.

FIGS. 12A and B. A, cDNA nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-CP-2.

FIGS. 13A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-CP-3.

FIGS. 14A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-CP-4.

FIGS. 15A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-CP-5.

FIGS. 16A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-SAA-1.

FIGS. 17A and B. A, nucleotide sequence; and B, amino acid sequenceencoded by nucleotide sequence for Na-SAA-2.

FIGS. 18A and B. Individual titers of BALB/c mice given the indicateddoses of Na-ASP-2/Alhydrogel® (80 mcg Alhydrogel®g) with and without 5mcg ODN 2006 in 50 mcL i.m. at days 0 and 20, with terminal bleeds atday 30 (log scale). A, arithmetic mean; B, geometric mean.

FIGS. 19A and B. A, Anti-Na-ASP-2 Specific IgG antibody responses inhumans immunized with Na-ASP-2, as determined by ELISA (undetectabletiters were arbitrarily assigned a titer of 50); B, proliferativeresponse of peripheral blood mononuclear cells from humans immunizedwith Na-ASP-2, after in vitro stimulation with Na-ASP-2.

FIG. 20. Ranking criteria for larval antigens for the human hookwormvaccine.

FIG. 21. Ranking criteria for adult antigens for the human hookwormvaccine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

It is an object of this invention to provide a bivalent human hookwormvaccine. The vaccine is effective at inducing an immune response inindividuals to whom it is administered, and administration results in areduction in symptoms of hookworm disease, e.g. worm burden, blood loss,etc.

The vaccine comprises: one or more L3 larval stage antigen and at leastone adult stage human hookworm antigen (e.g. Na-APR-1, Na-CP-2, Na-CP-3,Na-CP-4, Na-CP-5, or Na-GST-1) and one or more adjuvants. In someembodiments, the vaccine composition includes two or more adjuvants, oneof which mya be andaluminum-based adjuvant such as Alhydrogel®.

In a preferred embodiment of the invention, the antigens are Necatoramericanus antigens.

With respect to the one or more larval stage antigens that are used inthe vaccine, exemplary antigens are Na-ASP-2, Na-SAA-1, and Na-SAA-2,the sequences of which are found in FIGS. 1, 16 and 17, respectively.

With respect to the one or more adult stage antigens that may be used inthe vaccine composition, the following exemplary sequences arecontemplated: Na-APR-1 Shanghai strain (partial sequence 62-1351 bp) asdepicted in FIG. 2; Na-APR-1 Australia strain as depicted in FIG. 3;Na-APR-1 amino acid sequence (without signal) alignment between Shanghaiand Australia strains as depicted in FIG. 4; Pichia Optimized Na-APR-1(Na-APR-1-O) Sequence (based on Australia strain) as depicted in FIG. 5;Pichia Optimized Na-APR-1 (Na-APR-1-O) with Asp97 mutated to Ala97 asdepicted in FIG. 6; Pichia Optimized Na-APR-1 (Na-APR-1-O) with Asp284mutated to Ala284 as depicted in FIG. 7; Pichia Optimized Na-APR-1(Na-APR-1-O) with both Asp97/Asp284 mutated to Asp97/Ala284 as depictedin FIG. 8; Na-GST-1 as depicted in FIG. 9; Na-GST-2 as depicted in FIG.10; Na-GST-3 as depicted in FIG. 11; Na-CP-2 as depicted in FIG. 12;Na-CP-3 as depicted in FIG. 13; Na-CP-4 as depicted in FIG. 14; andNa-CP-5 as depicted in FIG. 15.

“Larval stage antigen” or “L3 larval stage antigen” refers to antigensthat are expressed during the L3 larval stage of the hookworm lifecycle. In some cases, such antigens may also be expressed during otherstages of the life cycle, i.e. the antigen may not be expressedexclusively in the larval stage. However, a “larval stage antigen” isexpressed at least in the L3 larval stage.

In preferred embodiments, Pichia optimized Na-APR-1 sequences are used,as described in numbers 4-7 above. Codon optimization enhances theefficiency of DNA expression vectors used in DNA vaccination byincreasing protein expression. The codon frequency of the foreign (i.e.hookworm) DNA embedded into the yeast expression vector may not beoptimal for adequate protein expression in the host resulting in lowlevel protein expression. A potential solution for the codon bias is tooptimize the codon sequences of a gene to suit the requirements of thehost without altering the original amino acid sequence of the proteinSee, for example, Jareborg N, Durbin R, ‘Alfresco—a workbench forcomparative genomic sequence analysis’, Genome Res 2000Aug;10(8):1148-57., 16; and Kim C H, Oh Y, Lee T H: Codon optimization forhigh level expression of human erythropoietin (EPO) in mammalian cells.Gene 199:293-301 (1997).

With respect to the adult stage GST antigen, three exemplary Na-GSTamino acid sequences, Na-GST-1, Na-GST-2, and Na-GST-3, are representedin FIGS. 9B, 10B and 11B, respectively, and nucleotide sequences thatencode these antigens are represented in FIGS. 9A, 10A and 10B,respectively.

With respect to the adult stage Na-CP antigens that are used in thevaccine, exemplary amino acid sequences of this antigen are representedin FIG. 12B (Na-CP-2), 13B (Na-CP-3), 14B (Na-CP-4), and 15B (Na-CP-5)and exemplary nucleotide sequences that encode these antigens arerepresented in FIGS. 12A, 13A, 14A and 15A, respectively.

With respect to the larval stage SAA antigens, exemplary nucleic acidsequences and encoded amino acid sequences of Na-SAA-1 and Na-SAA-2 aregiven in FIGS. 17 and 18, respectively.

Examples of antigens, their amino acid primary sequences, and nucleicacid sequences which encode them are given herein, and any combinationof the antigens depicted herein may be used in the practice of theinvention. However, those of skill in the art will recognize that manyvariants of the sequences presented herein may exist or be constructedwhich would also function as antigens in the practice of the presentinvention. For example, with respect to amino acid sequences, variantsmay exist or be constructed which display: conservative amino acidsubstitutions; non-conservative amino acid substitutions; truncation by,for example, deletion of amino acids at the amino or carboxy terminus,or internally within the molecule; or by addition of amino acids at theamino or carboxy terminus, or internally within the molecule (e.g. theaddition of a histidine tag for purposes of facilitating proteinisolation, the substitution of residues to alter solubility properties,the replacement of residues which comprise protease cleavage sites toeliminate cleavage and increase stability, the addition or eliminationof glycosylation sites, and the like, or for any other reason). Suchvariants may be naturally occurring (e.g. as a result of naturalvariations between species or between individuals); or they may bepurposefully introduced (e.g. in a laboratory setting using geneticengineering techniques). All such variants of the sequences disclosedherein are intended to be encompassed by the teaching of the presentinvention, provided the variant antigen displays sufficient identity tothe described sequences. Preferably, identity will be in the range ofabout 50 to 100%, or in the range of about 75 to 100%, or in the rangeof about 80 to 100%, or 85% to 100%, or 90% to 100%, or about 95% to100% of the disclosed sequences. The identity is with reference to theportion of the amino acid sequence that corresponds to the originalantigen sequence, i.e. not including additional elements that might beadded, such as those described below for chimeric antigens.

The invention also encompasses chimeric antigens, for example, antigenscomprised of the presently described amino acid sequences plusadditional sequences which were not necessarily associated with thedisclosed sequences when isolated but the addition of which conveys someadditional benefit. For example, such benefit may be utility inisolation and purification of the protein, (e.g. histidine tag, GST, andmaltose binding protein); in directing the protein to a particularintracellular location (e.g. yeast secretory protein); in increasing theantigenicity of the protein (e.g. KHL, haptens). All such chimericconstructs are intended to be encompassed by the present invention,provided the portion of the construct that is based on the sequencesdisclosed herein is present in at least the indicated level of homology.

Those of skill in the art will recognize that it may not be necessary toutilize the entire primary sequence of a protein or polypeptide in orderto elicit an adequate antigenic response to the parasite from which theantigen originates. In some cases, a fragment of the protein is adequateto confer immunization. Thus, the present invention also encompassesantigenic fragments of the sequences disclosed herein, and their use invaccine preparations. In general, such a fragment will be at least about10-13 amino acids in length. Those of skill in the art will recognizethat suitable sequences are often hydrophilic in nature, and arefrequently surface accessible.

Likewise, with respect to the nucleic acid sequences disclosed herein,those of skill in the art will recognize that many variants of thesequences may exist or be constructed which would still function toprovide the encoded antigens or desired portions thereof. For example,due to the redundancy of the genetic code, more than one codon may beused to code for an amino acid. Further, as described above, changes inthe primary sequence of the antigen may be desired, and this wouldnecessitate changes in the encoding nucleic acid sequences. In addition,those of skill in the art will recognize that many variations of thenucleic acid sequences may be constructed for purposes related tocloning strategy, (e.g. for ease of manipulation of a sequence forinsertion into a vector, such as the introduction of restriction enzymecleavage sites, etc.), for purposes of modifying transcription (e.g. theintroduction of promoter or enhancer sequences, and the like), or forany other suitable purpose. All such variants of the nucleic acidsequences disclosed herein are intended to be encompassed by the presentinvention, provided the sequences display about 50 to 100% identity tothe original sequence and preferably, about 75 to 100% identity, andmost preferably about 80 to 100% identity. The identity is withreference to the portion of the nucleic acid sequence that correspondsto the original sequence, and is not intended to cover additionalelements such as promoters, vector-derived sequences, restriction enzymecleavage sites, etc. derived from other sources.

In a preferred embodiment, the vaccine of the present invention includesan aluminum-based adjuvant such as the aluminum hydroxide adjuvantAlhydrogel® (available from Superfos and Brenntag Biosector) or thealuminum-containing adjuvant AS04 (available from GlaxoSmithKline). Inaddition, at least one additional adjuvant is also a component of thevaccine. Exemplary additional or second adjuvants include but are notlimited to the following:

1) AS03, a proprietary formulation manufactured by Glaxo Smith Klinethat contains an oil-in-water emulsion;2) AS02A, a proprietary formulation manufactured by Glaxo Smith Klinethat contains the same oil-in-water emulsion as in AS03, plus twoimmunostimulants “3D-MPL” and “QS-21”.AS03 and AS02A are described (under their original designations SBAS3and SBAS2, respectively) is Stoute et al NEJ M 1997 336:86-91. It isnoted that, AS02A and AS03 are designed to be used with the aluminumbased adjuvant AS04, also available from GlaxoSmithKline.3) A synthetic oligodeoxynucleotide adjuvant containing cytosine-guaninedinucleotides in particular base contexts or CpG motifs, (CpG ODN). Thisadjuvant is an immunomodulatory molecule and is available from Coley.4) Various lipid A derivatives. Lipid A is the portion oflipopolysaccharide that is known to be the primary component with regardto adjuvanticity and toxicity. Derivatives of lipid A have been producedin an attempt to retain the immunostimulatory activity of Lipid A yetreduce the toxicity. One such derivative, monophosphoryl lipid A (MPL,available from Chiron), has been shown to exhibit strong Th1 adjuvantactivity but with a considerably reduced toxicity compared to LPS. MPLhas adjuvant activity whether used alone, or in combination with otherimmunostimulants, such as CpG ODN, or aluminum hydroxide. Anothersynthetic lipid A derivative that is very similar to thelipopolysaccharide derivative lipid A monophosphoryl (MPL) by Chiron isavailable from the Infectious Disease Research Institute, Seattle, Wash.5) A publication by McCluskie and Weeratna (Infectious Disorders, 2001,1, 263-271) gives examples of several different adjuvant systems, eachof which may be employed in the practice of the present invention.

Examples of other suitable adjuvants include but are not limited toSeppic, Quil A, etc. Preferred adjuvants combinations are:Alhydrogel®+CpG 10103 and Alhydrogel®+synthetic lipid A.

The present invention provides compositions for use in eliciting animmune response against hookworm. The compositions may be utilized as avaccine against hookworm. By “eliciting an immune response” we mean thatan antigen stimulates synthesis of specific antibodies at a titer ofabout >1 to about 1×10⁶ or greater. Preferably, the titer is from about10,000 to about 1×10⁶ or more, as measured by enzyme LinkedImmunosorbent Assay (ELISA) or greater than 1,000 antibody units asdefined previously (Malkin et al., 2005a; 2005b). By “vaccine” we meanan antigen or antigen preparation that elicits an immune response thatresults in a decrease in hookworm burden of a least about 30% in anorganism in relation to a non-vaccinated (e.g. adjuvant alone) controlorganism. This work burden reduction has been calculated to restore achild's daily iron requirements that would otherwise be lost from amoderate (i.e. infections with between 2,000 and 4,000 hookworm eggs pergram of feces) infection with hookworm Preferably, however, the level ofthe decrease in hookworm burden would approach 50%, or more.

The present invention provides compositions for use in eliciting animmune response which may be utilized as a vaccine against hookworm. Thecompositions include a substantially purified recombinant hookwormantigen or variant thereof as described herein, and a pharmacologicallysuitable carrier. The preparation of such compositions for use asvaccines is well known to those of skill in the art. Typically, suchcompositions are prepared either as liquid solutions or suspensions,however solid forms such as tablets, pills, powders and the like arealso contemplated. Solid forms suitable for solution in, or suspensionin, liquids prior to administration may also be prepared. Thepreparation may also be emulsified. The active ingredients may be mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredients. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol and the like, or combinationsthereof. In addition, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and the like. In addition, the composition may contain otheradjuvants. If it is desired to administer an oral form of thecomposition, various thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders and the like may be added. The composition ofthe present invention may contain any such additional ingredients so asto provide the composition in a form suitable for administration. Thefinal amount of hookworm antigen in the formulations may vary. However,in general, the amount in the formulations will be from about 1-99%.

The present invention also provides methods of eliciting an immuneresponse to hookworm and methods of vaccinating a mammal againsthookworm. The methods generally involve identifying a suitable vaccinerecipient, and administering a composition comprising the hookwormantigens and adjuvants described herein in a pharmacologicallyacceptable carrier to the recipient. The vaccine preparations of thepresent invention may be administered by any of the many suitable meanswhich are well known to those of skill in the art, including but notlimited to by injection, orally, intranasally, by ingestion of a foodproduct containing the antigens, etc. In preferred embodiments, the modeof administration is subcutaneous or intramuscular. Patients with anexisting worm burden may be treated with a de-worming agent such asbenzimazole, and then be provided with the vaccine.

The present invention provides methods to elicit an immune response tohook worm and to vaccinate against hookworm in mammals. In oneembodiment, the mammal is a human.

Those of skill in the art will recognize that, in general, in order tovaccinate (or elicit an immune response in) a species of interest (e.g.humans) against hookworm, the antigen which is utilized will be derivedfrom a species of hookworm which parasitizes the species of interest.For example, in general, antigens from Necator americanus may bepreferred for the immunization of humans, and antigens from Ancylostomacanium may be preferred for the immunization of dogs. However, this maynot always be the case. For example, Ancylostoma canium is known toparasitize humans as well as its primary canine host. Further,cross-species hookworm antigens may sometimes be highly effective ineliciting an immune response in a non-host animal, i.e. in an animalthat does not typically serve as host for the parasite from which theantigen is derived. Rather, the measure of an antigen's suitability foruse in an immune-stimulating or vaccine preparation is dependent on itsability to confer protection against invasion and parasitization by theparasite as evidenced by, for example, hookworm burden reduction orinhibition of hookworm associated blood loss (e.g. as measured byhematocrit and/or hemoglobin concentration. For example, for use in avaccine preparation, an antigen upon administration results in areduction in worm burden of at least about 30%, preferably at leastabout 50%, and most preferably about 60 to about 70%.

EXAMPLES Example 1 Scoring System for Determining an Efficacious HumanHookworm Vaccine

A scoring system that incorporates essential criteria for determining anefficacious human hookworm vaccine has been developed (Table 1). Thecriteria include endpoints that focus on pathology (blood loss, wormburdens), transmission (faecal egg counts), ease of process development(known function/structure of protein) and immunoepidemiology(associations between immune responses and infection intensities innaturally exposed/infected cohorts). Once produced in soluble form,recombinant versions of the major L3 ES products were tested for vaccineefficacy in the canine and hamster models of infection.

TABLE 1 Ranking of candidate hookworm antigens based on seven majorcriteria, and grading of each criterion to allow a final score ofvaccine efficacy to be tallied. 1 2 3 5 6 Adult worm Adult worm Reduced4 Known Human 7 8 reduction reduction host blood EPG* function/ immuno-Protective Final Antigen (dog) (hamster)^(§) loss reduction structureepidemiology homologs Score Grading 0-5 0-5 0-4 0-4 0-2 0-3 0-2 ASP-2 23 1 3 2 3 2 16/25 (64%) APR-1 2 3 3 3 2  ND^(†) 1 14/22 (64%) CP-2 1 2 03 2 ND 1  9/22 (41%) GST-1 2 3 0 1 2 ND 1  9/22 (41%) 1 Reflectsquintiles of reduction in worm burdens in dogs compared to controls 2Reflects quintiles of reduction in worm burdens in hamsters compared tocontrols 3 Each grade reflects an increase of 0.5 g.dL-1 hemoglobinabove control group 4 Reflects tertiles of epg reduction compared tocontrols 5 Function or structure known in hookworm (grade of 2) or in arelated helminth (grade of 1) - enables biochemical assay development 6Association between antibody response and reduced epg in people (number= strength of association) 7 Protective homologs in other nematodes(grade of 2) or infectious agents (grade of 1) 8 Tally of scores fromeach category; ND—deduct from final score *EPG—eggs per gram of feces;^(§)B. Zhan, S. Xiao, J. Bethony, A. Loukas, P. Hotez, unpublished datausing N. americanus in the hamster model; ^(†)ND—Not Determined.

Based on this ranking system, recombinant antigens ASP-2, Ac-APR-1, GSTand CP-2 were selected as a lead vaccine candidates for further processdevelopment, cGMP manufacture and clinical testing.

Evidence that ASP-2 is a protective antigen in dogs (Ac-ASP-2) andhamsters (Ay-ASP-2) was published by the inventors in Bethony et al(2005) and Gould et al (2004); Mendez et al (2005), respectively. Humanimmunoepidemological evidence pointing to the protective effect of ASP-2antibodies was published in Bethony et al (2005). Evidence that Na-ASP-2is protective in hamsters is unpublished, while evidence thatanti-Na-ASP-2 antibodies inhibit hookworm larval penetration in vitrowas published by Goud et al (2005). Evidence that APR-1 is a protectiveantigen in dogs (Ac-APR-1) was published by the inventors in Loukas etal (2005). Evidence that Na-APR-1 is protective is also available, butunpublished. Evidence that GST is a protective antigen in dogs(Ac-GST-1) was published by the inventors in Zhan et al (2005). Evidencethat CP-2 is a protective antigen (Ac-CP-2) was published by theinventors in Loukas et al (2004).

Example 2 Human Clinical Trial with Recombinant Na-ASP-2

A double-blind, placebo-controlled, randomized dose-escalation Phase 1study was carried out to evaluate the safety, tolerability, andimmunogenicity of three intramuscular administrations of the Na-ASP-2hookworm vaccine in healthy adult volunteers. Thirty-six subjectsbetween the ages of 18 and 45 were randomized to receive a 0.5 mLinjection of either vaccine or saline placebo intramuscularly on studydays 1, 56 (week 8), and 112 (week 16). Enrolled subjects were dividedinto three dose cohorts of twelve subjects each. Within each dosecohort, three subjects were randomized to receive saline placebo andnine subjects were randomized to receive one of three doses of theNa-ASP-2 hookworm vaccine. Those randomized to receive vaccine weregiven 10, 50, or 100 μg of Na-ASP-2 in the first, second and third dosecohorts, respectively. Higher dose concentrations or additional (secondor third) injections were not administered until the effects of thepreceding dose concentration and injection had been evaluated. Subjectswere evaluated for adverse events, vital signs, blood chemistries,hematology, and urinalysis.

The cumulative safety data from this trial has demonstrated that thevaccine is both safe and immunogenic in healthy, hookworm-uninfectedadults, with mild to moderate injection-site tenderness, erythema,swelling and pruritus being the most commonly observed vaccine-relatedadverse events. Induration and warmth at the injection site occurredless frequently. All injection site reactions were considered mild ormoderate in severity and were typical of those observed withaluminum-adjuvanted vaccines administered intramuscularly. The frequencyof injection site reactions was not dose-dependent, and did not increasewith successive vaccinations. Unusual injection site reactions wereobserved in one male participant in the 10 μg dose group and in threefemale subjects in the 50 μg dose group after the second injection.These reactions were delayed erythematous reactions ranging in size from5 to 12 cm in diameter that started approximately 10 days after theinjection and lasted for 1 to 4 days, resolving without incident.Several vaccinated individuals also experienced mild to moderatesystemic adverse events including fever, headache and nausea. Novaccine-related serious adverse events occurred during the study, and noclinically-significant alternations in clinical laboratory parameterswere observed.

The Na-ASP-2 hookworm vaccine induced a significant antigen-specific IgGantibody response in a dose-dependent manner (FIG. 10): there was astatistically significant difference between the placebo and vaccinegroups starting as early as 14 days after the second injection whichremained through the 8 month follow-up time point after the thirdinjection.

Isotyping revealed that the induced antibody response consistedprimarily of IgG1, with a small component due to IgG4. No appreciableantigen-specific IgM, IgA or IgE responses were detected. Finally,significant antigen-specific cellular immune responses were alsoobserved, with increasing responses seen after successive injections ofvaccine (FIG. 11). While the invention has been described in terms ofits preferred embodiments, those skilled in the art will recognize thatthe invention can be practiced with modification within the spirit andscope of the appended claims. Accordingly, the present invention shouldnot be limited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

Example 3 Comparative Immunogenicity of Na-ASP-2/Alhydrogel® with andwithout ODN 2006 in BALB/c Mice

For each adjuvant tested, compatibility and stability studies wereundertaken to ensure that all individual components (antigen, adjuvant1, adjuvant 2, etc.) were compatible and that adequate stability wasachieved upon formulation. For Alhydrogel® based vaccine formulations towhich other adjuvants were added, this involved assays that test antigenbinding, conformation, and integrity over various periods of time and atdifferent temperatures.

With reference to FIGS. 13 A and B, Groups of 10 female BALB/c mice weregiven the indicated doses of Na-ASP-2/Alhydrogel® with or without 5micrograms ODN 2006, as indicated. Total antigen-specific IgG wasmeasured in the sera of each animal by indirect ELISA.

FIGS. 14 A and B show the geometric and arithmetic means, respectively,of the results. As can be seen, a comparative immunogenicity study ofNa-ASP-2/Alhydrogel® with and without ODN 2006 in BALB/c mice wasperformed. The results showed the ODN2006 boosts the immune response inBALB/c mice over that achieved with Na-ASP-2/Alhydrogel® alone, asdetermined by indirect ELISA that measure total antigen specific IgG(antibody).

REFERENCES FOR BACKGROUND AND EXAMPLES 1-3

-   Albonico M, Smith P G, Ercole E, Hall A, Chwaya H M, Alawi K S,    Savioli L, 1995. Rate of reinfection with intestinal nematodes after    treatment of children with mebendazole or albendazole in a highly    endemic area. Trans R Soc Trop Med. Hyg. 89:538-41.-   Albonico M, Bickle Q, Ramsan M, Montresor A, Savioli L,    Taylor M. 2003. Efficacy of mebendazole and levamisole alone or in    combination against intestinal nematode infections after repeated    targeted mebendazole treatment in Zanzibar. Bull World Health Organ.    81:343-52.-   Albonico M, Engels D, Savioli L. 2004. Monitoring drug efficacy and    early detection of drug resistance in human soil-transmitted    nematodes: a pressing public health agenda for helminth control.    Int J. Parasitol. 34:1205-10.-   Bethony J M, Loukas A, Smout M J, Mendez S, Wang Y, Bottazzi M E,    Zhan B, Williamson A L, Lustigman S, Correa-Oliveira R, Xiao S H,    Hotez P J. 2005. Antibodies against a secreted protein from hookworm    larvae reduce the intensity of infection in humans and vaccinated    laboratory animals. FASEB Journal 19: 1743-5.-   Bethony J, Chen J, Lin S, et al. Emerging patterns of hookworm    infection: influence of aging on the intensity of Necator infection    in Hainan Province, People's Republic of China. Clin Infect Dis    2002; 35: 1336-44.-   Bethony J, Brooker S, Albonico M, Geiger S, Loukas A, Diemert D,    Hotez P J. 2006. Soil-transmitted helminth infections: ascariasis,    trichuriasis, and hookworm. Lancet 367: 1521-32-   Chan M S. 1997. The global burden of intestinal nematode    infections—fifty years on. Parasitol Today 13:438-43.-   Cooper P J, Chico M, Sandoval C, et al. Human infection with Ascaris    lumbricoides is associated with suppression of the interleukin-2    response to recombinant cholera toxin B subunit following    vaccination with the live oral cholera vaccine CVD 103-HgR. Infect    Immun 2001; 69: 1574-80.-   Cooper P J, Espinel I, Wieseman M, et al. Human onchocerciasis and    tetanus vaccination: impact on the postvaccination antitetanus    antibody response. Infect Immun 1999; 67: 5951-7.-   DeSilva N, Brooker S, Hotez P, Montresor A, Engels D,    Savioli L. 2003. Soil-transmitted helminth infections: updating the    global picture. Trends in Parasitology 12: 547-51-   Elliott A M, Namujju P B, Mawa P A, et al. A randomised controlled    trial of the effects of albendazole in pregnancy on maternal    responses to mycobacterial antigens and infant responses to bacille    Calmette-Guerin (BCG) immunisation [ISRCTN32849447]. BMC Infect Dis    2005; 5: 115.-   Goud G N, Zhan B, Ghosh K, Loukas A, Hawdon J, Dobardzic A, Deumic    V, Liu S, Dobardzic R, Zook R C, Qun J, Liu Y Y, Hoffman L,    Chung-Debose D, Patel R, Mendez S, Hotez P J. 2004. Cloning, yeast    expression, isolation and vaccine testing of recombinant Ancylostoma    secreted protein 1 (ASP-1) and ASP-2 from Ancylostoma ceylanicum.    Journal of Infectious Diseases 189: 919-29.-   Goud G N, Bottazzi M E, Zhan B, Mendez S, Deumic V, Pleiskatt J, Liu    S, Wang Y, Bueno L, Fujiwara R, Samuel A, Ahn S Y, Solanki M, Asojo    O, Wen J, Saul A, Bethony J M, Loukas A, Roy M, Hotez P J. 2005.    Expression of the Necator americanus hookworm larval antigen    Na-ASP-2 in Pichia pastoris and purification of the recombinant    protein for use in human clinical trials. Vaccine 2005; 4754-64.-   Hotez P J, Ferris M T. The antipoverty vaccines. Vaccine 2006; 24:    5787-99.-   Hotez, P, Brooker S, Bethony J, Bottazzi M, Loukas A, Xiao S. 2004.    Hookworm Infection. New England Journal of Medicine 351: 799-807.-   Hotez P J, Bethony J, Bottazzi M E, Brooker S, Buss P. 2005.    Hookworm —“the great infection of mankind.” PLOS Medicine 2: e67    177-81.-   Hotez P J, Bethony J, Bottazzi M E, Brooker S, Diemert D, Loukas A.    2006a. New technologies for the control of human hookworm infection.    Trends in Parasitology 22: 327-31.-   Hotez P J, Molyneux D H, Fenwick A, Ottesen E, Ehrlich Sachs S,    Sachs J D. 2006b. Incorporating a rapid impact package for neglected    tropical diseases with programs for HUV/AIDS, tuberculosis, and    malaria. PLoS Medicine 3: e102.-   Kremer M, Miguel E. The illusion of sustainability. Center for    Global Development working paper, 2004.-   Loukas A, Bethony J M, Williamson A L, Goud G N, Mendez S, Zhan B,    Hawdon J M, Bottazzi M E, Brindley P J, Hotez P J. 2004. Vaccination    of dogs with recombinant cysteine protease from the intestine of    canine hookworms diminishes fecundity and growth of worms. Journal    of Infectious Diseases 189: 1952-61.-   Loukas A, Bethony J M, Mendez S, Fujiwara R T, Goud G N, Ranjit N,    Zhan B, Jones B, Bottazzi M E, Hotez P J. 2005. Vaccination with    recombinant aspartic hemoglobinase reduces parasite load and blood    loss after hookworm infection. PLoS Medicine 2: e295.-   Loukas A, Constant S L, Bethony J M. Immunobiology of hookworm    infection. FEMS Immunol Med Microbiol 2005; 43: 115-24.-   Malkin E M, Diemert D J, McArthur J H, Perreault J R, Miles A R,    Giersing B K, Mullen G F, Orcutt A, Muratova O, Awkal M, Zhou H,    Wang J, Stowers A, Long C A, Mahanty S, Miller L H, Saul A, Durbin    A H. 2005a. Infect. Immun. 73: 3677-85.-   Malkin E M, Durbin A P, Diemert D J, Sattabongkot J, Wu Y, Miura K,    Long C A, Lambert L, Miles A P, Wang J, Stowers A, Miller L H,    Saul A. 2005b. Phase 1 vaccine trial of Pvs 25H: a transmission    blocking vaccine for Plasmodium vivax malaria. Vaccine 23: 3131-8.-   Mendez S, Zhan B, Goud G, Ghosh K, Dobardzic A, Wu W H, Liu S,    Deumic V, Dobardzic R, Liu Y Y, Bethony J, Hotez P J. 2005. Effect    of combining the larval antigens Ancylostoma secreted protein 2    (ASP-2) and metalloprotease 1 (MTP-1) in protecting hamsters against    hookworm infection and disease caused by Ancylostoma ceylanicum.    Vaccine 23: 3123-30.-   Su Z, Segura M, Morgan K, Loredo-Osti J C, Stevenson M M. Impairment    of protective immunity to blood-stage malaria by concurrent nematode    infection. Infect Immun 2005; 73: 3531-9.-   World Health Organization. 2005. Deworming for Health and    Development, Report of the third global meeting of the partners for    parasite control, Geneva 29-30, November 2004.-   Zhan B, Liu S, Perally S, Fujiwara R, Brophy P, Liu Y Y, Feng J J,    Williamson A, Wang Y, Bueno L L, Mendez S, Goud G, Bethony J M,    Hawdon J M, Loukas A, Jones K, Hotez P J. 2005. Biochemical    characterization and vaccine potential of a heme binding glutathione    S transferase (GST) from the adult hookworm Ancylostoma caninum    Infection and Immunity 73: 6903-11.

Example 4 Hookworm Vaccine Antigens Screening with Necatoramericanus-Hamster Model 1. Introduction

Among the three major soil-transmitted nematodes, Ascaris lumbricoides,Ancylostoma duodenale/Necator americanus (hookworms), and Trichuristrichuria, hookworms are the most pathogenic because of their bloodfeeding behavior that directly causes blood loss and iron deficiencyanemia (de Silva et al, 2003; Bethony et al, 2006,). More seriously forchildren and women who have low iron stores, hookworm infection cancause retardation of physical and intellectual development (Bundy et al1995; Brooker et al, 1999; Hotez et al, 2006. 2004a).

More than 700 million people living in the developing countries oftropical and subtropical regions are estimated to be infected withhookworms. Hookworm infection causes more DALYs lost (1.8 million) thanany other helminthiases with the exception of lymphatic filariasis(Hotez et al, 2006. 2004a, Bethony et al, 2006). Mass chemotherapyremains a mainstay of hookworm control strategies (WHO 2002; Allen etal, 2002; Hotez et al, 2002). Indeed, repeated chemotherapy at regularintervals in high-risk groups is useful to keep a low morbidity, andwill frequently result in immediate improvement in child health anddevelopment (Bhargava et al, 2003; Stephenson et al, 1989) althoughcontinued used of anthelmintics is perhaps contributing to thedevelopment of anthelminthic resistance (Albonico et al, 2004).Unfortunately, the treated people, particularly, in highly endemicareas, soon become reinfected as early as 4-12 months after drugtreatment. Therefore, preventive vaccine against hookworm infectionbecomes an attractive alternative for hookworm control.

The major obstacle for developing human hookworm vaccine is the absenceof a suitable laboratory animal host to complete human hookworm's lifecycle (Hotez et al, 2003a,b, 2004b). Several laboratories have tried toinfect N. americanus in mice, dogs, guinea pigs, rabbits and hamsters(Timothy and Behnke, 1993, 1997; Nagahana et al, 1962; Yoshida et al,1960; Yoshida and Fukutome 1967; Sen, 1970; Sen and Seth, 1970; Sen andDeb, 1973). However, the efforts were not successful either due toinconsistent maintenance of the organism within the laboratory animals,the requirement for cortisone-immuno-suppression, or use of infantanimals. However, great progress was made by Xue and her colleagues (Xueet al, 2003a,b) in the Institute of Parasitic Diseases (IPD), ChineseCenter of Disease Control and Prevention (CCDCP) who successfullyadapted N. americanus to the Chinese golden hamster Mosocricetus auratuswithout the requirement for exogenous steroids or otherimmunosuppression, or the requirement to infect infant hamsters.Infection with the human hookworm N. americanus, originally obtainedfrom an infected patient living in Human Province, China, has beenestablished in the golden hamster Mosocricetus auratus for more than 100generations over a period of 26 years with no need of steroids (Xue etal, 2003). This model has been successfully used for testinganthelminthic drugs (Xue et al, 2005).

Several hookworm vaccine antigens have been tested with an Ancylostomacaninum-dog model or Ancylostoma ceylanicum-hamster model and some ofthem exhibited certain degrees of protection against A. caninum L3challenge with reduction of either adult worm burden or blood loss(Hotez, et al, 2003a; Goud et al, 2004; Mendez et al, 2005; Loukas etal, 2005, Bethony et al, 2005, Fujiwara (in press)). Among the vaccinestested, Na-ASP-2 is a leading antigen (Bethony et al, 2005; Goud et al,2005). However, these animal models are used to test vaccine antigensfrom animal hookworms such as A. caninum or A. ceylanicum. The effect ofsuch vaccines can be used to deduce or mimic the effect of humanhookworm homologues, but can not reflect completely the real pattern ofhuman hookworm. The Necator americanus-hamster model currently is theonly animal model for maintaining the species of human hookworms. Thishuman hookworm model was thus used to test various hookworm vaccinecandidates. The results showed that some of the antigens conferredprotective against symptoms of hookworm infection.

2. Materials and Methods 2.1 Hamsters

Male Chinese golden hamsters Mesocricetus auratus with an age of 7-8weeks were supplied by either Shanghai Institute of Biological Productsof the Chinese Ministry of Health or Shanghai Animal Center, ChineseAcademy of Sciences (SCXK(Hu) 2003-0003). The hamsters were housed ingroups of 10 in plastic cages. All animals had free access to water andcommercial rodent food purchased from Shanghai Shiling Biological andScientific Technique Corporation.

2.2 Vaccine Antigen and Adjuvant

Ten recombinant hookworm proteins derived either from N. americanus orA. caninum were used to test vaccine effect with the N.americanus-hamster model performed in the IPD. CCDCP. Na-ASP-2, a majorAncylostoma-secreted protein-2 secreted by stimulated infective larvaeof N. americanus, is a leading hookworm vaccine antigen. The recombinantNa-ASP-2 either with his-tag at C-terminal or without tag were expressedin the Pichia pastoris X-33 and purified with chromatography (Goud etal, 2005, Hawdon et al, 1999, Mendez et al, 2005).

Na-ASP-1 is another Ancylostoma-secreted protein secreted by stimulatedinfective larvae of N. americanus (Hawdon et al, 1996, Goud et al,2004). Ac-GST-1, a novel glutathione S-transferase produced by A.caninum adult worms, is a heme binding protein that is believed to beinvolved in the detoxification of heme derived from blood feeding (Zhanet al, 2005). Ac-CP-2 is a cathepsin-B cysteine protease from A. caninuminvolved in hemoglobin digestion of parasite (Harrop et al, 1995, Loukaset al, 2004). Na-CP-2 and Na-CP-4 are homologues of Ac-CP-2 cloned byscreening cDNA library of N. americanus with partial Ac-cp-2 cDNA(unpublished). Ac-APR-1 is a cathepsin D-like aspartic protease from A.caninum (Williamson et al, 2002, 2003; Loukas et al, 2005). Ac-MTP is anastacin-like metalloprotease secreted by the stimulated infective larvaeof A. caninum (Zhan et al, 2002, Williamson et al, 2006, Mendez et al,2005). Na-CTL is a C-type Lectin of N. americanus (Daub et al, 2000).Na-SAA-1 is a N. americanus orthologue of Ac-SAA-1, an immunodominantsurface-associated antigen from A. caninum (Zhan et al, 2004). Allrecombinant proteins were expressed in Pichia pastoris as solublesecretory proteins and purified with chromatography except for Na-SAA-1and Na-CP-2 that were expressed in E. coli. Recombinant Na-SAA-1 wassoluble and Na-CP-2 was insoluble and denatured in the 0.1% SDS.

The hookworm recombinant proteins were formulated with adjuvants ofeither Freund's, ASO₃ or Alhydrogel®. Complete and incomplete Freund'sadjuvants were obtained from Sigma (Saint Louis, Mo.). Twenty-five μg ofrecombinant protein was emulsified with 100 μl of complete Freund's foreach hamster for the first immunization and with incomplete Freund's forthe boost. ASO3 is a water-oil adjuvant (Stoute et al, 1997) kindlyprovided by GlaxoSmithKline (Rixensart, Belgium). Total volume of 100 μlof ASO3 was formulated with 25 μg of recombinant protein for eachhamster by mixing for 30 minutes at room temperature. Formulation ofantigen with Alhydrogel® was performed by mixing 25 μg of therecombinant protein with 25 μg of 2% Alhydrogel® in a total volume of200 μl for each hamster.

2.3 Vaccination

The dose of each vaccine given to each hamster was 25 μg recombinantprotein formulated with different adjuvants (Freund's, AS03 andAlhydrogel®) in a total volume of 200 μl. The vaccine was administratedsubcutaneously and booted twice with two weeks interval. Total of 10-26hamsters were immunized with one vaccine, the same number of hamsterswere injected with the same volume of adjuvant alone on the sameimmunization schedule as a control group. For Freund's adjuvant,complete Freund's adjuvant was used in the initial immunization,followed by two boosts with incomplete Freund's adjuvant.

2.4 Challenge with the Third-Stage Infective Larvae of N. americanus

The third stage infective larvae of N. americanus were collected fromcoprocultures of feces from hamsters infected with N. americanus larvae(Xue, 2003a, b). One week after the last immunization, the hamsters(vaccine and adjuvant control groups) were infected with fresh 150infective larvae subcutaneously under the skin of central abdomen.

2.5. Necropsy and Evaluation of vaccine effect

Twenty-five to twenty-eight days post challenge, all hamsters withvaccinated or control groups were sacrificed and the hookworms locatedin the small intestine were collected and counted. The mean worm burdenin each group was calculated. The differences between each vaccinatedgroup and the control group were analyzed by using Student t-test.

3. Results

3.1 Protective Immunity of rNa-ASP-2

In the first trial, the AS03 was used as adjuvant. In rNa-ASP-2 (withhis-tag) group, 26 hamsters were used and the mean worm burden was11.7±9.2, while 20.0±15.0 worms were found in the adjuvant controlgroup. The difference between the two groups was statisticallysignificant (P<0.05) (Table 2).

In the second trial, the protective effects of rNa-ASP-2 with his-tagand without his-tag were compared. However, the adjuvant was changed toAnhydrogel instead of SO₃. In this trial, the mean worm burden ofadjuvant group was 37.7±13.6, while those of rNa-ASP-2 (with his-tag)and rNa-ASP-2 (without his-tag) were 26.4±17.2 and 27.1±28.3,respectively.

The difference of mean worm burdens between the rNa-ASP-2 (with his-tag)group and control group was statistically significant with a wormreduction rate of 30.0%. No significant difference was seen in mean wormburdens between the rNa-ASP-2 without his-tag and control group becauseof large standard deviation appeared in the vaccine group (Table 2).

Overall, the mean worm reduction rate combining the three trials is31.8%, a statistically significant result when compared with theadjuvant only group.

TABLE 2 Protective immunity elicited by immunizing recombinant rNa-ASP-2in hamsters challenged with N. americanus L3 Vaccine mean Control meanWorm Vaccine worm ± SD worm ± SD reduction Trial# antigen (hamster#)(hamster#) rate(%) P value 1 rNa-ASP-2 11.7 ± 9.2 (26) 20.0 ± 15.0 (26)41.5 <0.05 (with his-tag) 2 rNa-ASP-2 26.4 ± 17.2 (20) 37.7 ± 13.6 (20)30.0 <0.05 (with his-tag) 3 rNa-ASP-2 27.1 ± 28.3 (20) 37.7 ± 13.6 (20)28.1 >0.05 (w/o his-tag) Total 21.7 ± 18.2 (66) 31.8 ± 14.0 (66) 31.8<0.053.2 Protective Immunity of rAc-GST-1

rAc-GST-1, the glutathione S-transferase-1 of A. caninum., wasformulated with the adjuvant Alhydrogel® for immunization of hamsters.The dosage of rAc-GST-1 used for immunization was 25 μg/hamster. In thefirst test, the mean worm burden in the rAc-GST-1 immunization group was15.7±9.8 which was less than that of 33.9±15.0 in the Alhydrogel® group,with a worm reduction of 53.7%. In the second test, the mean worm burdenin the rAc-GST-1 group was 16.7±6.6 which was similar to that of20.2±8.1 in the adjuvant group, with a worm reduction rate of 17.3%.Therefore, a third test was performed. The mean worm burden in therAc-GST-1 immunization group was significantly lower than that in thenonimmunized group with a worm reduction rate of 71.3%. When the resultsfrom the three tests were combined together for calculation, the meanworm burden in the immunization group was also lower than that of theadjuvant group, with a worm reduction rate of 48.4% (Table 3).

TABLE 3 Protective immunity elicited by immunizing recombinant rAc-GST-1in hamsters challenged with N. americanus L3 Vaccine mean Control meanWorm Vaccine worm ± SD worm ± SD reduction Trial# antigen (hamster#)(hamster#) rate (%) P value 1 rAc-GST-1 15.7 ± 9.8 (18) 33.9 ± 15.0 (20)53.7 <0.05 2 rAc-GST-1 16.7 ± 6.6 (19) 20.2 ± 8.1 (19) 17.3 >0.05 3rAc-GST-1  7.1 ± 7.8 (20) 24.6 ± 10.5 (21) 71.3 <0.01 Total 13.0 ± 9.0(57) 25.2 ± 13.0 (60) 48.4 <0.013.3 Protective Immunity of rNa-CP-2

Na-CP-2, cysteine protease-2 of N. americanus, was cloned by screeningN. americanus L3 cDNA library with Ac-CP-2. The recombinant protein wasexpressed in E. coli. In the first trial, the mean worm burden inhamsters immunized with rNa-CP-2 was significantly lower than that inadjuvant group with worm reduction rate of 42%. In the repeat test, thedifference of mean worm burdens between rNa-CP-2 group and adjuvantgroup was not significant. When the results of the two tests werecombined together, the mean worm burden in rNa-CP-2 group wassignificantly lower than that in the adjuvant group (Table 4)

TABLE 4 Protective immunity elicited by immunizing recombinant rNa-CP-2in hamsters challenged with N. americanus L3 Vaccine mean Control meanWorm Vaccine worm ± SD worm ± SD reduction Trial# antigen (hamster#)(hamster#) rate (%) P value 1 rNa-CP-2 26.6 ± 23.1 (20) 45.9 ± 27.9 (21)42.0 <0.05 2 rNa-CP-2 31.8 ± 15.0 (20) 36.7 ± 25.6 (12) 13.4 >0.05 Total29.2 ± 19.1 (40) 42.4 ± 26.7 (33) 31.1 <0.053.4 Protective immunity of rAc-APR-1

Ac-APR-1, aspartic protease-1 secreted by A. caninum adult worm, is ahemoglobinase for worm to digest host blood hemoglobin as resource ofnutrition, therefore a good target for developing vaccine. Each hamsterwas immunized with 25 ug of recombinant Ac-APR-1 precipitated with 25 ulof 2% Alhydrogel®. After being boosted twice with the same formulationof recombinant Ac-APR-1, hamsters of vaccine and adjuvant group werechallenged with 150 N. americanus L3. The mean worm burden of vaccinatedgroup is 20.4±11.4 that is significantly lower than that from adjuvantcontrol group (36.7±25.6) (Table 5)

TABLE 5 Protective immunity elicited by immunizing recombinant rAc-APR-1in hamsters challenged with N. americanus L3 Vaccine mean Adjuvant meanWorm Vaccine worm ± SD worm ±SD reduction antigen Adjuvant (hamster#)(hamster#) rate (%) P value rAc-APR-1 Alhydrogel ® 20.4 ± 11.4 (16) 36.7± 25.6 (12) 44.4.0 <0.05Conclusion: These results show that vaccination with recombinantAc-APR-1 resulted in a marked decrease in worm burden after L3challenge. Ac-APR-1 thus affords protection against challenge withhookworm larvae to vaccinated hamsters.

3.5 Other Hookworm Vaccine Trials

Other 6 hookworm antigens (rNa-ASP-1, rAc-CP-2, rNa-CTL, rAc-MTP,rNa-CP-4, and rNa-SAA-1) were tested for their protective immunity inthe N. americanus-hamster model. The result showed no protective effectfor all antigens listed above for hamsters to resist infection with N.americanus L3 (Table 6).

TABLE 6 Hookworm recovery from hamsters after being immunized withdifferent hookworm recombinant proteins. Vaccine mean Adjuvant mean WormVaccine worm SD worm ±SD Reduction antigen Adjuvant (hamster#)(hamster#) rate (%) P value rNa-ASP-1 Freund's 29.1 ± 13.3 (8) 18.4 ±14.5 (8) 0 — rAc-CP-2 Freund's 35.7 ± 19.1 (9) 18.4 ± 14.5 (8) 0 —rNa-CTL Freund's 25.4 ± 15.5 (8) 18.4 ± 14.5 (8) 0 — rAc-MTPAlhydrogel ® 32.4 ± 24.4 (20) 45.9 ± 27.9 (21) 29.4 >0.05 rNa-CP-4Alhydrogel ® 20.5 ± 11.9 17.4 ± 13 (20) 0 — rNa-SAA-1 Alhydrogel ® 19.2± 15.2 17.4 ± 13 (20) 0 —

REFERENCES FOR EXAMPLE 4

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Example 5 Further Evaluations

An innovative scoring system has been used to select larval antigens foruse in the practice of the invention. The criteria were based on fivecriteria including an evaluation of the antigen in preclinical studiesto 1) reduce host worm burdens, 2) reduce host blood loss, 3) reducefecal egg counts, and 4) for antibody to inhibit larval invasion invitro. The fifth criterion was 5) whether there are known orthologuesthat protect in veterinary vaccines and the sixth criterion was 6) thefeasibility and ease of expression, yield and stability. Other factorsunder consideration included a known function and mechanism of action,association with reductions in risk of acquiring heavy hookworminfection in endemic setting, and immunoepidemiology. The results arepresented in tabular form in FIGS. 20 and 21. By these rankings, ASP-2(a L3 secreted antigen) and SAA-2 (a L3 surface antigen) emerged as thetwo lead candidate larval antigens and APR-1 and GST-1 emerged as thelead candidate adult antigens, with CP-2/3 (cysteine protease) and Cys(cystatin) as viable back-up antigens.

1. A vaccine composition, comprising, at least one larval-stage hookwormantigen; at least one adult-stage hookworm antigen; an aluminum-basedadjuvant; and a second adjuvant.
 2. The vaccine composition of claim 1,wherein said larval-stage hookworm antigen is selected from the groupconsisting of Na-ASP-2 and Na-SAA-2, or antigenic fragments thereof. 3.The vaccine composition of claim 1, wherein said adult-stage hookwormantigen is selected from the group consisting of Na-APR-1, Na-GST,Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or antigenic fragments thereof. 4.The vaccine of claim 3, wherein said Na-APR-1 is Pichia optimizedNa-APR-1, or an antigenic fragment thereof.
 5. The vaccine compositionof claim 1, wherein said aluminum-based adjuvant is Alhydrogel®.
 6. Thevaccine composition of claim 1, wherein said second adjuvant is selectedfrom the group consisting of: CpG and Synthetic lipid A.
 7. The vaccinecomposition of claim 1, wherein said aluminum-based adjuvant and saidsecond adjuvant are combined together.
 8. A method for vaccinating apatient in need thereof against hookworm infections, comprising the stepof administering to said patient a vaccine composition comprising, atleast one larval-stage hookworm antigen; at least one adult-stagehookworm antigen; an aluminum-based adjuvant; and a second adjuvant. 9.The method of claim 8, wherein said larval-stage hookworm antigen isselected from the group consisting of Na-ASP-2 and Na-SAA-2, orantigenic fragments thereof.
 10. The method of claim 8, wherein saidadult-stage hookworm antigen is selected from the group consisting ofNa-APR-1, Na-GST, Na-CP-2, Na-CP-3, Na-CP-4, Na-CP-5, or antigenicfragments thereof.
 11. The method of claim 10, wherein said Na-APR-1 isPichia optimized Na-APR-1, or an antigenic fragment thereof.
 12. Themethod of claim 8, wherein said aluminum-based adjuvant is Alhydrogel®.13. The method of claim 8, wherein said second adjuvant is selected fromthe group consisting of: CpG and Synthetic lipid A.
 14. The method ofclaim 8, wherein said aluminum-based adjuvant and said second adjuvantare combined together.
 15. The method of claim 8, further comprising thestep of administering a deworming agent to said patient.
 16. A hookwormvaccine comprising a hookworm larval stage antigen; an hookworm adultstage antigen; and one or more adjuvants.
 17. A method for vaccinating apatient in need thereof against hookworm infections, comprising the stepof administering to said patient a vaccine composition comprising, ahookworm larval stage antigen; an hookworm adult stage antigen; and oneor more adjuvants.